An advanced model to analyze CD4 T cell reactions to antibody and BCR V regions Project Summary This is an R03 proposal to generate an antibody construct and a corresponding transgenic mouse for genetic analyses of T cell reactions to V region peptides derived from antibodies and B cell antigen receptors (BCR). CD4 T cells make a living by interacting with B cells that present antigenic peptides in MHC II. While some of these peptides come from foreign antigen, others are neoantigens generated in the BCR through the processes of V/D/J gene recombination in the bone marrow and somatic hypermutation in the periphery. Neoantigenic BCR peptides in MHC II open a potential avenue of T cell help to B cells, in violation of the principle of linked recognition. We refer to this as the receptor presentation avenue of help. Neoantigenic Ab peptides may also activate CD4 T cells at sites of Ab deposition where APC gather Ab through Fc receptors or surface lectins. At present, we have only a rudimentary understanding of how the immune system regulates potentially dangerous interactions between CD4 T cells and B cells or other APC that are mediated by BCR and Ab peptides. As an example, IgG is ostensibly tolerogenic;yet therapeutic mAb sometimes elicit an endogenous neutralizing antibody response with a potential clinical health hazard. We are pursuing adoptive transfer studies to reveal the consequences of T cell reactions to BCR- and Ab- derived peptides using a model based in mice of the strain A genetic background. Our most recent results indicate that the "receptor presentation" avenue of T help to B cells is subject to immunoregulation. T-B interactions in the GC that are mediated by BCR-derived peptides impede GC participation and memory B cell development and ultimately induce a refractory state in the T cells. Efforts to dissect this novel form of immunoregulation and to explore the issue of Ab immunogenicity versus tolerogenicity, would be greatly enhanced if we could utilize the available library of genetically altered C57BL/6 (B6) mice. Accordingly, we would like to develop a corresponding model in the B6 strain so that genetic alterations can be introduced into adoptively transferred B cells, T cells or recipient mice. A B6 model would also enable us to genetically analyze CD4 T cell reactions to an antibody V region peptide in adoptive recipients of T cells that are injected with mAb carrying the cognate peptide. To this end, we propose to engineer an Ig;light chain construct that contains a sequence encoding a peptide called 3K, which is immunogenic for CD4 T cells in the context of by I-Ab (Aim 1). This will enable production of hapten-specific (NP) mAb of various isotypes for planned future studies of T cell responses to 3K;Ig, immune complexes thereof and glycan modified forms of 3K;Ig. For these studies, we would use a currently available 12TCR Tg mouse as a source of 3K-specific CD4 T cells. In Aim 2, we propose to engineer a modified 3K;genomic construct with a GFP reporter and generate a corresponding B6 transgenic mouse. This mouse will be crossed with a knock-in mouse (B1-8) that carries a complementary targeted heavy chain from an Ig;NP-specific mAb to produce a mouse with NP-specific 3K;B cells. These B cells can then be used with 3K-specific T cells in future co-adoptive transfer genetic studies of receptor presentation. The products of this work will enable a genetic dissection of mechanisms that regulate T cell responses to Ig V region peptides in the BCR or secreted antibody. The anticipated results from this model will advance our understanding of immunoregulation in the face of neoantigens that frequently arise in BCR and Ab and are presented to CD4 T cells on a regular basis during immunity. Abbreviations ANA: Anti-nuclear antibody BAC: Bacterial artificial chromosome BCR: B cell antigen receptor CFP: Cyan fluorescent protein GFP: Green fluorescent protein GC: Germinal Center NP: (4-hydroxy-3-nitro-phenyl)acetyl SHM: Somatic hypermutation PUBLIC HEALTH RELEVANCE: T lymphocytes that react against peptides derived from antibodies are potential health hazards in a variety of situations. They may induce patient responses against therapeutic monoclonal antibodies;they may induce organ inflammation when directed against antibodies that deposit in the organ, and they may stimulate B cells to produce antibodies against self-tissues in systemic autoimmune diseases such as systemic lupus erythematosus (SLE). This project aims to develop a unique state-of-the-art-model that will enable us to apply the power of genetics to reveal how T cells react to antibody peptides and mechanisms that regulate potential adverse outcomes. The information obtained from this model will provide a sound basis for the rational design of therapies to control or prevent inflammatory and autoimmune diseases in which antibodies and B cells play a significant role.